Molecular Basis for Lytic Bacteriophage Resistance in Enterococci

Abstract

The human intestine harbors diverse communities of bacteria and bacteriophages. Given the specificity of phages for their bacterial hosts, there is growing interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. A significant barrier to such therapies is the rapid development of phage-resistant bacteria, highlighting the need to understand how bacteria acquire phage resistance in vivo Here we identify novel lytic phages in municipal raw sewage that kill Enterococcus faecalis, a Gram-positive opportunistic pathogen that resides in the human intestine. We show that phage infection of E. faecalis requires a predicted integral membrane protein that we have named PIPEF (for phage infection protein from E. faecalis). We find that PIPEF is conserved in E. faecalis and harbors a 160-amino-acid hypervariable region that determines phage tropism for distinct enterococcal strains. Finally, we use a gnotobiotic mouse model of in vivo phage predation to show that the sewage phages temporarily reduce E. faecalis colonization of the intestine but that E. faecalis acquires phage resistance through mutations in PIPEF Our findings define the molecular basis for an evolutionary arms race between E. faecalis and the lytic phages that prey on them. They also suggest approaches for engineering E. faecalis phages that have altered host specificity and that can subvert phage resistance in the host bacteria.

Importance: Bacteriophage therapy has received renewed attention as a potential solution to the rise in antibiotic-resistant bacterial infections. However, bacteria can acquire phage resistance, posing a major barrier to phage therapy. To overcome this problem, it is necessary to understand phage resistance mechanisms in bacteria. We have unraveled one such resistance mechanism in Enterococcus faecalis, a Gram-positive natural resident of the human intestine that has acquired antibiotic resistance and can cause opportunistic infections. We have identified a cell wall protein hypervariable region that specifies phage tropism in E. faecalis Using a gnotobiotic mouse model of in vivo phage predation, we show that E. faecalis acquires phage resistance through mutations in this cell wall protein. Our findings define the molecular basis for lytic phage resistance in E. faecalis They also suggest opportunities for engineering E. faecalis phages that circumvent the problem of bacterial phage resistance.

FIG 1

Genome organization of lytic phages φVPE25 and φVFW. Whole-genome alignments were performed using MAFTT version 1.3 (61). Open reading frames for φVPE25 and φVFW were determined using RAST version 2.0, and the resulting data were imported into Geneious 6.0.6. Modular gene organization based on predicted function is color coded. Vertical lines indicate regions with a high degree of nucleotide heterogeneity between φVPE25 and φVFW. Transmission electron microscopy revealed that φVPE25 and φVFW are noncontractile tailed siphophages.

FIG 2

φVPE25 and φVFW DNA is modified at cytosine residues. (A) Structure of methylation and glycosylation modifications that occur at cytosine residues in DNA. Cytosine can be methylated in the form of a single methyl group (5mC) or hydroxyl-methylated (5hmC). 5hmC can be converted to a glucose-linked cytosine (5ghmC) by glucosyltransferase. (B) DNA sequencing analysis of sodium bisulfite-treated E. faecalis OG1RF genomic DNA or φVPE25 and φVFW genomic DNA. Unmodified cytosine in E. faecalis genomic DNA is converted to uracil after bisulfite treatment and when sequenced appears as thymidine. φVPE25 and φVFW genomic DNA resists bisulfite conversion, confirming cytosine modification. Dots indicate that these nucleotides match those in the consensus sequence. (C) Restriction endonuclease digestion of genomic DNA from φVPE25, φVFW, and E. faecalis V583. Incomplete digestion by PvuRts1I may be due to a minimum number of glycosylation sites or to inefficient DNA cleavage.

FIG 3

EF0858 encodes PIP_EF, promotes phage infection, and harbors a hypervariable region. (A) Cross streak and plaque assays using φVPE25 show the susceptibility or resistance profiles of E. faecalis V583 and the isogenic PIP_EF deletion strain BDU50. Introduction of the pLZPIP plasmid, which contains the entire open reading frame of PIP_EF restores phage infectivity of E. faecalis BDU50. pLZ12 is the empty vector. (B) Topological cartoon of E. faecalis V583 PIP_EF generated using TOPCONS (62). The cartoon depicts PIP_EF as an integral membrane protein that spans the membrane six times. The ~160-amino-acid variable region of PIP_EF is represented as the black box with a black netting pattern in the large extracellular domain. (C) Pairwise amino acid sequence alignments of 19 PIP_EF homologs were performed using Geneious 6.0.6. The N and C termini are indicated.

FIG 4

A variable region in PIP_EF determines phage tropism in E. faecalis. (A) Schematic of PIP_EF from E. faecalis V583. A variable region covering ~160 amino acids is located in the center of the PIP_EF coding sequence. (B) Both φVPE25 cross streak and plaque assays show that truncations in the PIP_EF variable region abolish phage infectivity regardless of location. The deletions (ΔA, ΔB, and ΔC) correspond to the colored amino acids highlighted in the magnified area of the variable region shown in panel A. (C) Susceptibility profiles of 19 E. faecalis strains for phages φVPE25 and φVFW. (D) Clustering of PIP_EF variable region amino acid alignments from 19 strains of E. faecalis. Strains cluster according to their susceptibility patterns as determined in panel C. These strains are indicated by color coding as follows: strains sensitive to killing by both phages (black), strains sensitive to only φVPE25 (blue), and strains sensitive only to φVFW (red). Strains can be further grouped into five specific clades based on PIP_EF variable region amino acid identity. (E) Representative clade-specific mutation frequencies for phages φVPE25 and φVFW. NT, not tested (due to natural resistance to the phage of interest).

FIG 5

PIP_EF swapping alters phage tropism. (A) Using the E. faecalis strain E1Sol, which is naturally resistant to infection by φVPE25, both cross streak and plaque assays showed that E1Sol can acquire φVPE25 susceptibility by expressing the E. faecalis V583 PIP_EF gene (pLZPIP). Single-copy replacement of the E. faecalis E1Sol PIP_EF homolog with V583 PIP_EF in the E1Sol chromosome also confers φVPE25 sensitivity (PIP_V583). A chimera of E. faecalis E1Sol PIP_EF and the variable region of V583 PIP_EF show that the PIP_EF variable region determines phage tropism (pLZEV). (B and C) Plaquing efficiency of φVPE25 (B) and φVFW (C) on E. faecalis V583, E1Sol, and PIP_V583 transgenic E1Sol strains. The value that was significantly different (P < 0.01) by Student’s t test from the value for E. faecalis V583 is indicated by an asterisk. (D) Cross streak assay showing that expression of E. faecalis V583 PIP_EF from plasmid pPBPIP can confer φVPE25 sensitivity on E. faecium strains 1,141,733 and Com12, but not strain Com15.

FIG 6

PIP_EF promotes DNA entry, but not initial phage adsorption. (A to C) Initial phage adsorption to E. faecalis cells was measured by determining the percentage of phages remaining in the supernatant after the addition of various E. faecalis strains. NB, no bacteria added. (D to F) Southern blotting was performed using an φVPE25 whole-genome probe on DNA isolated from whole cells infected with φVPE25. (D) E. faecalis V583 compared to the isogenic PIP_EF mutant strain BDU50. (E) φVPE25 replication can be restored in strain BDU50 when PIP_EF is provided in trans. (F) The variable region of strain V583 is sufficient to allow φVPE25 DNA entry into E. faecalis, because φVPE25 DNA can replicate in strain E1Sol only if the strain expresses a PIP_EF chimera carrying the V583 PIP_EF variable region (pLZEV) in the large extracellular facing domain. pLZ12 is the empty vector control.

FIG 7

In vivo phage predation selects for E. faecalis PIP_EF mutants. Germfree C57BL6/J mice were orally inoculated with E. faecalis V583 followed by an oral treatment of φVPE25. (A) Fecal burden of E. faecalis from mice with and without phage treatment over a 9-day period. Each symbol represents the value for an individual mouse, and the short black bar represents the mean of the group of mice. The means that are significantly different (P < 0.001) by Student’s t test are indicated by a bar and asterisk. (B) φVPE25 particle numbers from gnotobiotic mouse feces as determined by a plaque assay. (C) Percentage of φVPE25-resistant E. faecalis clones isolated from gnotobiotic mice following phage treatment. The percentage of φVPE25 resistance was calculated by determining the number of phage-resistant isolates by cross streaking and then dividing the number of resistant isolates by the total number of isolates acquired at each time point and multiplying by 100. Symbols: ♦, treated with φVPE25; ●, not treated with phage.